Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K–Akt signaling

Phosphoinositide-3 kinase (PI3K)/Akt signaling is activated by growth factors such as insulin and epidermal growth factor (EGF) and regulates several functions such as cell cycling, apoptosis, cell growth, and cell migration. Here, we find that Kank is an Akt substrate located downstream of PI3K and a 14-3-3–binding protein. The interaction between Kank and 14-3-3 is regulated by insulin and EGF and is mediated through phosphorylation of Kank by Akt. In NIH3T3 cells expressing Kank, the amount of actin stress fibers is reduced, and the coexpression of 14-3-3 disrupted this effect. Kank also inhibits insulin-induced cell migration via 14-3-3 binding. Furthermore, Kank inhibits insulin and active Akt-dependent activation of RhoA through binding to 14-3-3. Based on these findings, we hypothesize that Kank negatively regulates the formation of actin stress fibers and cell migration through the inhibition of RhoA activity, which is controlled by binding of Kank to 14-3-3 in PI3K–Akt signaling

Development of Pathological Diagnostics of Human Kidney Cancer by Multiple Staining Using New Fluorescent Fluolid Dyes

New fluorescent Fluolid dyes have advantages over others such as stability against heat, dryness, and excess light. Here, we performed simultaneous immunostaining of renal tumors, clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, acquired cystic disease-associated RCC (ACD-RCC), and renal angiomyolipoma (AML), with primary antibodies against Kank1, cytokeratin 7 (CK7), and CD10, which were detected with secondary antibodies labeled with Fluolid-Orange, Fluolid-Green, and Alexa Fluor 647, respectively. Kank1 was stained in normal renal tubules, papillary RCC, and ACD-RCC, and weakly or negatively in all other tumors. CK7 was positive in normal renal tubules, papillary RCC, and ACD-RCC. In contrast, CD10 was expressed in renal tubules and clear cell RCC, papillary RCC, AML, and AC-RCC, and weakly in chromophobe RCC. These results may contribute to differentiating renal tumors and subtypes of RCCs. We also examined the stability of fluorescence and found that fluorescent images of Fluolid dyes were identical between a tissue section and the same section after it was stored for almost three years at room temperature. This indicates that tissue sections can be stored at room temperature for a relatively long time after they are stained with multiple fluorescent markers, which could open a door for pathological diagnostics

Kank attenuates actin remodeling by preventing interaction between IRSp53 and Rac1

In this study, insulin receptor substrate (IRS) p53 is identified as a binding partner for Kank, a kidney ankyrin repeat–containing protein that functions to suppress cell proliferation and regulate the actin cytoskeleton. Kank specifically inhibits the binding of IRSp53 with active Rac1 (Rac1G12V) but not Cdc42 (cdc42G12V) and thus inhibits the IRSp53-dependent development of lamellipodia without affecting the formation of filopodia. Knockdown (KD) of Kank by RNA interference results in increased lamellipodial development, whereas KD of both Kank and IRSp53 has little effect. Moreover, insulin-induced membrane ruffling is inhibited by overexpression of Kank. Kank also suppresses integrin-dependent cell spreading and IRSp53-induced neurite outgrowth. Our results demonstrate that Kank negatively regulates the formation of lamellipodia by inhibiting the interaction between Rac1 and IRSp53

Estrogenic Activity of Coffee Constituents

Here, the constituents of coffee with estrogenic activity are summarized by a comprehensive literature search, and their mechanisms of action for their physiological effects are discussed at the molecular and cellular levels. The estrogenic activity of coffee constituents, such as acids, caramelized products, carbohydrates, lignin, minerals, nitrogenous compounds, oil (lipids), and others, such as volatile compounds, was first evaluated by activity assays, such as animal tests, cell assay, ligand-binding assay, protein assay, reporter-gene assay, transcription assay, and yeast two-hybrid assay. Second, the health benefits associated with the estrogenic coffee constituents, such as bone protection, cancer treatment/prevention, cardioprotection, neuroprotection, and the improvement of menopausal syndromes, were summarized, including their potential therapeutic/clinical applications. Inconsistent results regarding mixed estrogenic/anti-estrogenic/non-estrogenic or biphasic activity, and unbeneficial effects associated with the constituents, such as endocrine disruption, increase the complexity of the effects of estrogenic coffee constituents. However, as the increase of the knowledge about estrogenic cell signaling, such as the types of specific signaling pathways, selective modulations of cell signaling, signal crosstalk, and intercellular/intracellular networks, pathway-based assessment will become a more realistic means in the future to more reliably evaluate the beneficial applications of estrogenic coffee constituents

DNA Microarray‐Based Screening and Characterization of Traditional Chinese Medicine

The application of DNA microarray assay (DMA) has entered a new era owing to recent innovations in omics technologies. This review summarizes recent applications of DMA‐based gene expression profiling by focusing on the screening and characterizationof traditional Chinese medicine. First, herbs, mushrooms, and dietary plants analyzed by DMA along with their effective components and their biological/physiological effects are summarized and discussed by examining their comprehensive list and a list of representative effective chemicals. Second, the mechanisms of action of traditional Chinese medicine are summarized by examining the genes and pathways responsible for the action, the cell functions involved in the action, and the activities found by DMA (silent estrogens). Third, applications of DMA for traditional Chinese medicine are discussed by examining reported examples and new protocols for its use in quality control. Further innovations in the signaling pathway based evaluation of beneficial effects and the assessment of potential risks of traditional Chinese medicine are expected, just as are observed in other closely related fields, such as the therapeutic, environmental, nutritional, and pharmacological fields

Estrogen-like activity and dual roles in cell signaling of an Agaricus blazei Murrill mycelia-dikaryon extract

Agaricus blazei (A. blazei) Murrill mycelia-dikaryon has attracted the attention of scientists and clinicians worldwide owing to its potential for the treatment of cancer. However, little is known about its effect on other pathologies. This study sought to extend the potential medical usefulness of A. blazei for preventing vascular damage and to unravel its mechanism of action. The A. blazei extract showed estrogen-like activity in both gene expression profiling and a luciferase assay. Indeed, the extract inhibited oxidized low-density lipoprotein-stimulated activation of Erk1/2, Akt and p38 in HUVECs and macrophage-derived TIB-67 cells. Moreover, the extract enhanced transcription of the glutathione peroxidase 3 (GPX3), alpha-synuclein (SNCA) and endothelial nitrogen-oxide synthase (eNOS) genes. Furthermore, atherosclerotic lesions in rabbits were reduced by intake of A. blazei powder. Therefore, A. blazei may be useful for preventing atherosclerosis via dual roles in cell signaling, suppression of macrophage development and the recovery of endothelial cells from vascular damage